ABSTRACT
The products of the proto-oncogenes c-fos and c-jun play important roles in cell growth control, differentiation, and malignant transformation. Purified oncogenic proteins are essential tools in cell growth control studies. Here we describe the production of mouse cFos and cJun oncoproteins in Sf9 insect cells using the baculovirus expression system. The mouse c-fos cDNA was subcloned into two different baculovirus expression vectors, namely pVL1392 and pVLMH6, to generate, respectively, nonfusion and His-fusion cFos oncoproteins in insect cells. The products were characterized by immunofluorescence, immunoblotting, immunoprecipitation, and ability to bind to the in vitro translated JunB protein to form the AP-1 complex. A His-cJun fusion protein was also produced in insect cells using the pVLMH6 baculovirus vector. Coexpression of cFos and cJun in insect cells yielded functional AP-1 complexes as judged from gel retardation assays. The results point to the possibility of using insect cells for structural and functional studies of different Fos/Jun AP-1 complexes.
Subject(s)
Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-jun/genetics , Transcription Factor AP-1/metabolism , Animals , Baculoviridae , Base Sequence , Blotting, Southern , Cloning, Molecular , DNA/metabolism , DNA, Complementary/isolation & purification , Humans , Mice , Molecular Sequence Data , Protein Binding , Recombinant Fusion ProteinsABSTRACT
The use of the baculovirus system to produce recombinant proteins is based on the high level of protein production and the possibility to obtain, in Spodoptera frugiperda insect cells, recombinant proteins with the post-translational modifications found in the native proteins. Here we describe the isolation and characterization of a recombinant baculovirus containing the mouse c-fos gene. The c-fos cDNA was subcloned into the pVL1392 baculovirus transfer vector. The recombinant plasmid (pVL1392.fos) was introduced into Sf9 insect cells by co-transfection with viral wild-type DNA. Upon selection and characterization of a viral recombinant clone, Sf9 cells were infected with this virus stock and the cFos protein expression was detected by immunological methods using an anti-cFos polyclonal antiserum.
Subject(s)
Baculoviridae/metabolism , Proto-Oncogene Proteins c-jun/isolation & purification , Animals , DNA Restriction Enzymes/analysis , Mice , Sequence Analysis, DNA , Spodoptera , TransfectionABSTRACT
The use of the baculovirus system to produce recombinant proteins is based on the high level of protein production and the possibility to obtain, in Spodoptera frugiperda insect cells, recombinant proteins with the post-translational modifications found in the native proteins. Here we describe the isolation and characterization of a recombinant baculovirus containing the mouse c-fosgene. The c-fos cDNA was subcloned into the pVL1392 baculovirus transfer vector. The recombinant plasmid (pVL1392.fos) was introduced into Sf9 insect cells by co-transfection with viral wild-type DNA. Upon selection and characterization of a viral recombinant clone, SF9 cells were infected with this virus stock and the cFos protein expression was detected by immunological methods using an anti-cFos polyclonal antiserum